Towards On-Demand E. coli-Based Cell-Free Protein Synthesis of Tissue Plasminogen Activator

Stroke is the leading cause of death with over 5 million deaths worldwide each year. About 80% of strokes are ischemic strokes caused by blood clots. Tissue plasminogen activator (tPa) is the only FDA-approved drug to treat ischemic stroke with a wholesale price over $6000. tPa is now off patent although no biosimilar has been developed. The production of tPa is complicated by the 17 disulfide bonds that exist in correctly folded tPA. Here, we present an Escherichia coli-based cell-free protein synthesis platform for tPa expression and report conditions which resulted in the production of active tPa. While the activity is below that of commercially available tPa, this work demonstrates the potential of cell-free expression systems toward the production of future biosimilars. The E. coli-based cell-free system is increasingly becoming an attractive platform for low-cost biosimilar production due to recent developments which enable production from shelf-stable lyophilized reagents, the removal of endotoxins from the reagents to prevent the risk of endotoxic shock, and rapid on-demand production in hours.

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Endotoxin-Free E. coli- Based Cell-Free Protein Synthesis: Pre-Expression Endotoxin Removal Approaches for on-Demand Cancer Therapeutic Production

Biotechnology Journal ◽

10.1002/biot.201800271 ◽

2018 ◽

Vol 14 (3) ◽

pp. 1800271 ◽

Cited By ~ 21

Author(s):

Kristen M. Wilding ◽

John P. Hunt ◽

Joshua W. Wilkerson ◽

Parker J. Funk ◽

Rebecca L. Swensen ◽

...

Keyword(s):

Protein Synthesis ◽

Free Protein ◽

E Coli ◽

On Demand ◽

Cell Free Protein Synthesis

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Sequence Specific Modeling ofE. coliCell-Free Protein Synthesis

10.1101/139774 ◽

2017 ◽

Cited By ~ 2

Author(s):

Michael Vilkhovoy ◽

Nicholas Horvath ◽

Che-Hsiao Shih ◽

Joseph A. Wayman ◽

Kara Calhoun ◽

...

Keyword(s):

Protein Synthesis ◽

Fluorescent Protein ◽

Regulation Of Gene Expression ◽

Training Data ◽

Model Parameters ◽

Free System ◽

Free Protein ◽

E Coli ◽

Constraint Based Modeling ◽

Cell Free Protein Synthesis

AbstractCell-free protein synthesis (CFPS) is a widely used research tool in systems and synthetic biology. However, if CFPS is to become a mainstream technology for applications such as point of care manufacturing, we must understand the performance limits and costs of these systems. Toward this question, we used sequence specific constraint based modeling to evaluate the performance ofE. colicell-free protein synthesis. A coreE. colimetabolic network, describing glycolysis, the pentose phosphate pathway, energy metabolism, amino acid biosynthesis and degradation was augmented with sequence specific descriptions of transcription and translation and effective models of promoter function. Model parameters were largely taken from literature, thus the constraint based approach coupled the transcription and translation of the protein product, and the regulation of gene expression, with the availability of metabolic resources using only a limited number of adjustable model parameters. We tested this approach by simulating the expression of two model proteins: chloramphenicol acetyltransferase and dual emission green fluorescent protein, for which we have training data sets; we then expanded the simulations to a range of additional proteins. Protein expression simulations were consistent with measurements for a variety of cases. The constraint based simulations confirmed that oxidative phosphorylation was active in the CAT cell-free extract, as without it there was no feasible solution within the experimental constraints of the system. We then compared the metabolism of theoretically optimal and experimentally constrained CFPS reactions, and developed parameter free correlations which could be used to estimate productivity as a function of protein length and promoter type. Lastly, global sensitivity analysis identified the key metabolic processes that controlled CFPS productivity and energy efficiency. In summary, sequence specific constraint based modeling of CFPS offered a novel means toa prioriestimate the performance of a cell-free system, using only a limited number of adjustable parameters. While we modeled the production of a single protein in this study, the approach could easily be extended to multi-protein synthetic circuits, RNA circuits or the cell free production of small molecule products.

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Development and comparison of cell-free protein synthesis systems derived from typical bacterial chassis

Bioresources and Bioprocessing ◽

10.1186/s40643-021-00413-2 ◽

2021 ◽

Vol 8 (1) ◽

Author(s):

Liyuan Zhang ◽

Xiaomei Lin ◽

Ting Wang ◽

Wei Guo ◽

Yuan Lu

Keyword(s):

Protein Synthesis ◽

Protein Production ◽

Influential Factors ◽

Competent Cell ◽

Free Protein ◽

Application Range ◽

E Coli ◽

Short Time ◽

Cell Free Protein Synthesis

AbstractCell-free protein synthesis (CFPS) systems have become an ideal choice for pathway prototyping, protein production, and biosensing, due to their high controllability, tolerance, stability, and ability to produce proteins in a short time. At present, the widely used CFPS systems are mainly based on Escherichia coli strain. Bacillus subtilis, Corynebacterium glutamate, and Vibrio natriegens are potential chassis cells for many biotechnological applications with their respective characteristics. Therefore, to expand the platform of the CFPS systems and options for protein production, four prokaryotes, E. coli, B. subtilis, C. glutamate, and V. natriegens were selected as host organisms to construct the CFPS systems and be compared. Moreover, the process parameters of the CFPS system were optimized, including the codon usage, plasmid synthesis competent cell selection, plasmid concentration, ribosomal binding site (RBS), and CFPS system reagent components. By optimizing and comparing the main influencing factors of different CFPS systems, the systems can be optimized directly for the most influential factors to further improve the protein yield of the systems. In addition, to demonstrate the applicability of the CFPS systems, it was proved that the four CFPS systems all had the potential to produce therapeutic proteins, and they could produce the receptor-binding domain (RBD) protein of SARS-CoV-2 with functional activity. They not only could expand the potential options for in vitro protein production, but also could increase the application range of the system by expanding the cell-free protein synthesis platform.

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